Human monoclonal antibodies to the islet cell antigen IA-2

ABSTRACT

The invention concerns human monoclonal antibodies to the islet cell antigen IA-2, a process for their production, the use of human monoclonal antibodies in a method for detecting antibodies to IA-2, a method for detecting antibodies to the islet cell antigen IA-2 and a method for detecting the islet cell antigen IA-2 in a sample.

The invention concerns human monoclonal antibodies to the islet cellantigen IA-2, a process for their production, the use of humanmonoclonal antibodies in a method for detecting antibodies to IA-2, amethod for detecting antibodies to the islet cell antigen IA-2 and amethod for detecting IA-2.

Type I insulin-dependent diabetes mellitus (IDDM) is due to anautoimmune destruction of the insulin-producing β cells of the pancreas.The development of autoantibodies to β cell antigens precedes thedevelopment of a clinically diagnosable diabetes. These autoantibodiesare sensitive markers for identifying the preclinical phase of thedisease. Antibodies that react with the β cells in the islets ofLangerhans are also frequently found in newly diagnosed diabeticpatients. The autoantibodies as a whole are also referred to as isletcell antibodies (ICA).

ICA are sensitive and specific markers for the prognosis and diagnosisof human IDDM. Previously characterized islet cell antigens to whichautoantibodies are formed include insulin (Palmer et al. 1983, Science222, 1337-1339), glutamate decarboxylase (GAD, Bakkeskov et al. 1990,Nature 347, 151-156), carboxypeptidase H (Castano et al. 1991, J. Clin.Endocrinol. Metab. 73, 1197-1201) islet cell antigen ICA 69 (Pietropaoloet al. 1993, J. Clin. Invest. 92, 359-371) and the antigen IA-2 which isalso referred to as ICA512 (Solimena et al. 1996, EMBO

Up to now it has not been possible to clarify whether autoantibodiesthat are directed towards β cell antigens contribute directly to thedevelopment of the disease or whether the occurrence of theautoantibodies is a phenomenon that occurs after the destruction of theβ cells.

However, according to present knowledge the occurrence of autoantibodiescorrelates with the development of diabetes.

It has been shown that especially autoantibodies to IA-2 occur in mostof the newly diagnosed IDDM patients and that the IA-2-specificautoantibodies are associated with a rapid progression of the diabetesdisease. In addition IA-2-specific autoantibodies appear to be morespecific for IDDM than GAD autoantibodies and moreover occur lessfrequently in other autoimmune diseases without IDDM (Roll and Ziegler1997, Exp. Clin. Endocrinol. Diabetes 105, 1-14).

The autoantigen IA-2 is a transmembrane protein that has a segment thatcrosses the membrane and a cytoplasmic domain (IA-2ic) which containsthe epitopes for antibody binding (Solimena et al. 1996, EMBO J. 15,2102-2114). IA-2 is an intrinsic membrane protein of secretory vesiclesthat is expressed in peptide secreting endocrine cells and in neuroneswhich contain neurosecretory vesicles. IA-2 has a significant homologyto IA-2β which is also known as phogrin. IA-2β is a transmembraneprotein like IA-2, but unlike IA-2 it is primarily expressed in β cells.IA-2β and IA-2 are proteins of the receptor type and both belong to theclass of protein tyrosine phosphatases (Roll and Ziegler 1997, supra).

In the prior art ICA (islet cell antibodies) are determined for thedetection of IDDM by measurements on pancreatic tissue sections usingindirect immunofluorescence. In this method the autoantibodies in thesample to be examined that bind to the islet cell structures aredetected by fluorescent-labelled antibodies that are specific for humanIgG. However, these ICA measurements are technically very complicatedand difficult to standardize since the results obtained with differentpancreatic tissues from different donors varies greatly.

In the prior art autoantibodies to IA-2 and GAD are also determined bysimple radioligand binding assays in serum samples. These assays use invitro translated, radioactively labelled antigens (Dittler, J. et al1998, Diabetes 47, 592-597). In order to prepare radioactively labelledantigens, the cDNA of the respective antigen is transcribed in vitrousing a rabbit reticulocyte lysate. The mRNA is then translated in thepresence of radioactively labelled amino acids (usually labelled with³⁵S, sulphur-35). Binding of the autoantibodies to the labelled antigenis detected by means of the radioactive signal of the labelled antigenafter separation of the antigen-antibody complex from the free antigenby for example filtration or solid phase binding.

Although these detection methods can be partially automated, they havethe major disadvantage that one has to work with radioactivity whichrequires laborious and expensive precautionary measures. The labellingefficiencies for the antigen that can be achieved by in vitrotranslation vary greatly from batch to batch. In addition the labelledantigens have a very short shelf life due to radiolysis and the shorthalf-life of sulphur-35.

A diagnostic test for the direct detection of IA-2-specificautoantibodies that can be carried out simply, rapidly and in anautomated manner has previously not been described in the prior art.This is in particular due to the fact that standardized IA-2-specificauto-antibodies have previously not been available. Hitherto onlyhigh-titre sera from IDDM patients were used as a standard orcalibration material. The disadvantage of this material is that suchserum is not available in an unlimited amount and thus batch andpatient-dependent variations in the antibody content of the respectivestandard sera occur. Hence there is no comparability betweenexperimental results from different laboratories.

The object was therefore to provide human monoclonal antibodies whichspecifically recognize the islet cell antigen IA-2 and to provide adiagnostic test procedure for the quantitative detection of theIA-2-specific autoantibodies by means of a standard curve which at leastpartially overcomes the disadvantages of the prior art.

The object is achieved by human monoclonal antibodies which specificallyreact with islet cell antigen IA-2. These for example include theantibodies which are produced by the cell line IA-2, 96-3-1, depositedat the DSMZ (“Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH”, Braunschweig, Germany) on 13.08.1998 under the number DSMACC2365.

The invention therefore concerns human monoclonal antibodies to theislet cell antigen IA-2. These antibodies are preferably of the IgGisotype, particularly preferably of the IgG1 isotype.

It is known in the prior art that human autoantibodies can be producedagainst the islet cell antigen GAD (glutamate decarboxylase) (EP-A-0 499176). The described method comprises the following steps: Immortalizinghuman lymphocytes of prediabetics or diabetics, treating the culturesupernatant of the immortalized cells (by EBV transformation) with aconjugate of antibodies to human Fcγ and a label, subsequent treatmentwith human immunoglobulin, incubation with immobilized human pancreaticislet cells or immobilized GAD, identification of an immortalized humancell culture which produces an antibody to pancreatic islet cells bydetermining the label bound to the immobilized islet cells or to theimmobilized GAD, isolation of a human immortalized cell which producesthis antibody, proliferation of this immortalized cell and isolation ofthe monoclonal antibody produced by these cells.

However, the method described in EP-A-0 499 176 does not allow theproduction of human monoclonal antibodies to IA-2. The first difficultyalready becomes apparent in the selection of the donor lymphocytes. Itis not possible to use any donor lymphocytes, but rather the lymphocytesmust be derived from selective prediabetics or diabetics with highIA-2-specific serum antibody titres.

The antibody titres are determined by a radioligand binding assay. Inthis assay the DNA coding for IA-2 is transcribed in vitro by areticulocyte lysate and translated in the presence of ³⁵S-methionine.Subsequently a few microlitres (2-5 μl have proven to be suitable)patient serum is incubated with the radioactively labelled IA-2 and theimmune complexes are separated from free antigen over protein-ASepharose. The radioactivity bound to the protein-A Sepharose isdetermined in a scintillation counter. The measured cpm are convertedinto arbitrary units by means of a selected high titre patient serum.For example it was defined that 1000 cpm corresponds to 100 U in thepatient serum used in this study. This patient serum is used as anarbitrary standard for the determinations. Only lymphocytes whose donorshad a titre of more than 80 U were used for the transformations sinceIA-2-positive primary cultures were only discovered for these donors.

Moreover it proved to be advantageous to carry out a preselection forIgG-producing B lymphocytes. In peripheral blood there are ca. 10 timesmore IgM-producing B lymphocytes than IgG-producing B cells. On theother hand the relevant autoantibodies to IA-2 are of the IgG subtype(Zhang et al, 1997, Diabetes 46, 40-43). The relevant B cellsubpopulation can be enriched 10-fold by isolating the membraneIgG-positive B lymphocytes. These are isolated by labelling the human Blymphocytes with an antibody from the mouse that is specific for humanIgG and subsequently binding magnetic beads that are coated with sheepanti-mouse IgG. The labelled cells can be positively selected from thecell suspension by applying a magnet.

Furthermore problems occurred in the culture of the immortalizedlymphocytes since the immortalized IA-2-specific B cells only had a lowproliferation rate and were frequently overgrown by unspecific butrapidly growing immortalized B cells. It turned out that theproliferation of the immortalized IA-2-specific B cell lines can begreatly improved by adding growth factors such as IL-6 or IL-10. Anadditional problem was that the immortalized B cell lines secretefactors which adversely affect the growth of the cell lines. Thesefactors include above all TGF-beta, IF-gamma and TNF-alpha. Removal ofthese inhibitory factors by frequently changing the culture mediumresults in a higher transformation rate and a more rapid growth of theEBV-transformed B cell lines.

A crucial factor for successful cloning (see below) of the resultingEBV-transformed B lymphocytes is to introduce limiting amounts of Blymphocytes into the individual wells (wells of the microtitre plate)right from the beginning. The prior art recommends several thousandpurified peripheral mononuclear cells per well (Peyron, E. et al. 1994,J. Immunology 153, 1333-1339; Madec. A.-M. et al 1996, J. Immunology156, 3541-3549). It surprisingly turned out that no more than 400IgG-positive cells/well can be used for the primary transformation. Thetransformation rate is about 1 out of 80 B cells, i.e. a maximum of 5different clones grow per well. This increases the probability that onecan isolate the relevant clones in the subsequent single cell cloning inwhich only a few EBV-transformed B cell clones grow (often only 1-2%).It is also surprising that most of the positive primary wells can beisolated at an even lower seeding density which can be interpreted tomean that already too many clones are formed with 400 B cells per welland consequently clones that grow more slowly are suppressed by rapidlygrowing cells.

After a growth period of ca. 2 weeks the culture supernatants of theprimary cell cultures are then tested for the production ofIA-2-specific antibodies for example by means of an ELISA test onimmobilized IA-2. Screening for IA-2-specific antibodies by means of thecytoplasmic domain of IA-2 which is also referred to as IA-2ic hasproven to be particularly suitable according to the invention.

So-called feeder cells have to be added in the subsequent single cellcloning for the stabilization of the cell lines since EBV-transformed Bcell lines cannot survive at a low cell density (<25 per well).Autologous (derived from the same donor) or allogenic (derived from adifferent donor) peripheral blood lymphocytes irradiated with 4000 radare used as feeder cells.

The cloning efficiency can be decisively improved by removing cytotoxicT lymphocytes (CD8-positive cells) from the feeder cell population. TheCD8 cells are preferably removed by immunomagnetic separation. For thisthe peripheral blood lymphocytes are for example incubated with magneticmicrobeads to which monoclonal antibodies to the human CD8 antigen arecoupled. The labelled cells are removed by applying a magnet, theremaining cells are irradiated and used at a concentration of20,000-50,000 per well.

It has also proven to be advantageous for the production of theantibodies according to the invention to carry out a quality check ofthe feeder cells. It was found that the growth-promoting function ofthese feeder cells varies greatly from donor to donor. The feeder cellsof some donors even had an inhibitory effect on growth. Hence a majorimprovement was to firstly examine each new batch of feeder cells on anestablished monoclonal EBHV-transformed B cell line (MICA 5) as a testcell line for their suitability for the cloning procedure and to sortout “harmful” feeder batches. For this purpose single cell clonings ofMICA 5 were carried out on irradiated blood lymphocytes from differentdonors. The cloning efficiency was determined after ca. 3 weeks bymicroscopic determination of the number of growth wells. Only thosefeeder cells were used which enabled a cloning efficiency of at least20%.

The immortalization step can be carried out by transformation with EBV(Epstein Barr virus) known to a person skilled in the art. Thistransformation is preferred according to the invention. The mostfrequently described method in the literature for EBV transformation(see Ifversen, P. et al 1993, Hum. Antibod. Hybridomas 4, 115-123) is toincubate B lymphocytes with EBV for 2-3 hours to allow virus uptake.Afterwards the cells are washed to remove the virus. However, it wassurprisingly found that the transformation rate could be increased bynot washing out the virus but instead to incubate it together with the Bcells during the entire incubation period up to the first change ofmedium (after ca. 7 days).

However, the immortalization can also be carried out by fusion withsuitable myeloma cells. It is also conceivable that the monoclonalantibodies to IA-2 according to the invention could be produced by theso-called phage display method (Nissim, A. et al. 1994, EMBO J. 13, 3,692-698). In this method the mRNA is isolated directly from thelymphocytes of the IDDM patients. The immunoglobulin genes can beamplified (for example by means of the polymerase chain reaction) fromthe cDNA prepared in this manner. The genes produced in this manner canin turn be expressed in a phage library as Fab or single chain Fv fromwhich the phages binding to IA-2 can be isolated.

It was not possible to produce human monoclonal antibodies to the isletcell antigen IA-2 until the difficulties described above had beenovercome.

Hence a preferred subject matter of the invention are monoclonalantibodies that bind to IA-2 and which are produced by the cell lineIA-2, 96-3-1, deposited on 13.08.1998 at the DSMZ (“Deutsche Sammlungvon Mikroorganismen und Zellkulturen GmbH”, Mascheroder Weg 1b, D-38124Braunschweig, Germany) under the number DSM ACC2365. The cell line DSMACC2365 is also a subject matter of the invention.

Antibodies are also a subject matter of the invention which can bind toIA-2 in an equivalent manner to those produced by the cell line IA-2,96-3-1 (DSM ACC2365). The term “can bind in an equivalent manner” refersto antibodies in which there is a detectable epitope overlap with thedefined known antibody. This epitope overlap can be easily detected withthe aid of a competitive test system. For example an enzyme immunoassayis used to examine the extent to which an antibody competes with theknown antibody for binding to a defined antigen or to a defined epitope(for example IA-2ic). For this immobilized IA-2ic antigen is for exampleincubated with the known monoclonal antibody which carries a label andwith an excess of the antibody under examination. It can then be easilydetermined to what extent the examined antibody can displace the definedantibody from binding to the antigen by detection of the bound label. Ifthere is a displacement of at least 50% with a 10⁵-fold excess, then anepitope overlap is present.

The invention additionally concerns human monoclonal antibodies to theislet cell antigen IA-2 which can be obtained by the process steps ofimmortalizing human lymphocytes from prediabetics or diabetics with highserum antibody titres (>80 U) to IA-2, culturing the immortalizedlymphocytes with growth factors while simultaneously removing inhibitoryfactors by frequently changing the medium, detecting the IA-2-specifichuman monoclonal antibodies in the culture supernatant preferably bymeans of ELISA, cloning the human immortalized cell line which producesthis antibody in the presence of feeder cells which contain no cytotoxicT lymphocytes, proliferating this immortalized cell optionally with theaddition of growth factors, and isolating the monoclonal antibodyproduced by this clone.

The invention also concerns a process for the production of humanmonoclonal antibodies which specifically react with the islet cellantigen IA-2 comprising the steps immortalizing human lymphocytes fromprediabetics or diabetics with high serum antibody titres (>80 U/ml) toIA-2, culturing the immortalized lymphocytes with growth factors whilesimultaneously removing inhibitory factors by frequently changing themedium, detecting the IA-2-specific human monoclonal antibodies in theculture supernatant preferably by means of ELISA, cloning the humanimmortalized cell line which produces this antibody in the presence offeeder cells which contain no cytotoxic T lymphocytes, proliferatingthis immortalized cell optionally with the addition of growth factors,and isolating the monoclonal antibody produced by this clone.

The individual steps of the process are carried out as described in theprevious sections.

The term “monoclonal antibody” in the sense of the invention isunderstood to include all antibody fragments in addition to the intactimmunoglobulins. These for example include Fab, Fab′ or F(ab)′₂fragments. If the term “antibody” is not supplemented by the words“monoclonal” or “polyclonal”, then it means both types of antibodiesi.e. chimeric constructs and all fragments listed above.

The IA-2-specific monoclonal antibodies according to the invention reactspecifically with IA-2 and they preferably react with the cytoplasmicpart of IA-2 the so-called IA-2ic. Hence the invention also concernsantibodies to IA-2 which react specifically with the cytoplasmic part ofIA-2 the so-called IA-2ic. The process steps described above are usedanalogously to produce these antibodies.

An ELISA test is preferably used to identify the IA-2-specificmonoclonal antibodies. At least 1000 primary wells have to be tested foreach donor in order to find anti-IA-2-specific EBV-transformed B celllines. Such an extensive screening cannot be carried out with the verylaborious RIA of the prior art. The very high sample throughput can onlybe achieved by developing an ELISA. This semiautomatic ELISA enablesseveral thousand culture supernatants to be tested per day and henceallows the discovery of the very seldom event of an IA-2-positiveprimary well.

In the ELISA the streptavidin-coated microtitre plates are coated withIA-2-biotin or IA-2ic-biotin. Subsequently the coated plates areincubated with various dilution steps of human sera from prediabetics orestablished diabetics. Defined amounts of a purified human IA-2-specificantibody are incubated concurrently on the same microtitre plate.Afterwards the plates are washed and a peroxidase-labelled sheepanti-human-Fcγ-specific antibody conjugate is added to detect boundanti-IA-2 antibodies. Bound IA-2-specific antibodies are detected by acolour reaction with ABTS® (azino-di-[3-ethylbenzthiazoline sulfonate(6)], catalogue No. 756 407, Boehringer Mannheim GmbH Germany). Thecontent of anti-IA-2 antibodies in patient sera can be deduced from theabsorbances of the standard curve taking into consideration the dilutionfactor.

In order to prepare the antigen IA-2ic which is used in the ELISA, theIA-2ic gene was amplified from an islet cell-specific cDNA using thefollowing primers which have been published by Payton et al. (1995) inJ. Clin. Invest. 96, 1506-1511 : 5′-ATGCAGCAAGACAACGAGCGCCTG-3′ and5′-TCACTGGGGCAGGGCCTTGAG-3′

The amplification products were cloned into a pin point vector andexpressed in E. coli as soluble, biotinylated fusion protein. The fusionprotein was purified on monomeric avidin-Sepharose. The biotinylatedIA-2ic was bound to streptavidin-coated microtitre plates, incubatedwith the IA-2-specific monoclonal antibodies according to the inventionand bound antibody was detected by a peroxidase-labelled anti-human IqGconjugate. In order to exclude the possibility that the antibodies weredirected against the biotinylated amino-terminal domain of the fusionprotein, the biotin binding domain of the fusion protein (Tag protein)was also expressed alone and tested in the ELISA. The IA-2-specificantibodies did not recognize the Tag protein. The IA-2-specificantibodies were also tested in a RIA. For this the DNA for IA-2ic wastranscribed and translated in vitro in the presence of ³⁵S-methionine,incubated with the IA-2-specific antibodies and the immune complexeswere immobilized by adding protein A Sepharose. The radioactivity in theimmunoprecipitates was determined by liquid scintillation counting.

The present invention also includes a method for detecting humanantibodies or autoantibodies to the islet cell antigen IA-2 in a sample.All formats familiar to a person skilled in the art come intoconsideration as test formats and an indirect ELISA test is preferred.It has proven to be suitable to contact purified native or recombinantIA-2 antigen or IA-2ic antigen with the sample such that the sampleantibodies can bind specifically to the antigen. If the antigen isprovided with a group that can bind to a solid phase such as biotin,then the immune complex can be subsequently immobilized on astreptavidin-coated solid phase. The antigen can also already bedirectly or indirectly bound to the solid phase when it is incubatedwith the sample. After separation of the solid from the liquid phase thesample antibodies are preferably detected by binding a labelled antibodywhich is directed against the Fc part of human antibodies, generally theFc part of human IgG and subsequently measuring the label. All labelsfamiliar to a person skilled in the art can be used as the label, forexample enzymes such as peroxidase, haptens such as digoxigenin,fluorescent dyes or substances capable of electrochemi-luminescence orchemiluminescence.

A competitive test format is also conceivable in which the IA-2 orIA-2ic antigen is bound directly or indirectly to a solid phase and adefined concentration of a labelled inventive human monoclonal antibodyto IA-2 or IA-2ic is added as a receptor and incubated with the antigen.If the sample is added simultaneously or subsequently, the sampleantibodies and labelled receptor antibodies compete with one another forbinding to the antigen. After separating the solid from the liquidphase, the label is determined in one or both phases. A low signal ofthe label on the solid phase indicates a high concentration of sampleantibodies.

Comparison of the resulting sample measurements with measured values fora series of standards that have been previously determined, enablequantification of the sample antibodies. Defined concentrations of theinventive monoclonal antibodies to IA-2 or IA-2ic are used in such aseries of standards.

All body fluids familiar to a person skilled in the art can be used assamples for detecting antibodies to IA-2. These for example includewhole blood, serum or plasma, urine and saliva.

A further subject matter of the invention is the use of a humanmonoclonal antibody to IA-2 or IA-2ic as a standard or as a receptor ina method for determining antibodies to an islet cell antigen, preferablyto the islet cell antigen IA-2.

A further subject matter of the invention is the use of a humanmonoclonal antibody to the islet cell antigen IA-2 for isolating theislet cell antigen IA-2. In order to isolate the islet cell antigenIA-2, the inventive antibodies can be coupled to a solid phase bymethods known to a person skilled in the art.

Subsequently the sample containing IA-2 is incubated with the antibodiesbound to the solid phase and the other components are separated.Cleavage of the immune complex between the antibody and antigen forexample by a high salt concentration and subsequent elution enable theantigen to be obtained in a pure form.

The invention also concerns anti-idiotypic antibodies whose antigenbinding site corresponds to the structure of the antigen IA-2 or IA-2ic.Such an anti-idiotypic antibody can be obtained by immunization with ahuman antibody to IA-2 according to the invention, immortalizing thespleen cells of the immunized animals, cloning those immortalized cellswhich produce antibodies that bind to the binding region of theIA-2-specific antibodies and isolating the antibodies produced by theseclones by known methods.

Another subject matter of the invention is a method for detecting IA-2in a sample which is characterized in that at least one monoclonalantibody according to the invention is used for this as the bindingpartner. The test is preferably carried out as a sandwich ELISA. Anantibody to IA-2 which can be an antibody according to the invention isused as a binding partner for this which is bound to a solid phase bymethods known to a person skilled in the art (for example viabiotin/streptavidin). The IA-2 present in the sample binds to theantibody that is bound to the solid phase. The bound IA-2 is detected bymeans of a further binding partner which carries a label. The furtherbinding partner is also preferably an antibody and also bindsspecifically to IA-2 but recognizes a different epitope to thatrecognized by the binding partner that is bound to the solid phase. Thelabelled binding partner can be a monoclonal antibody according to theinvention if the antibody bound to the solid phase recognizes anotherepitope. All labels familiar to a person skilled in the art can be usedas the label. These for example include enzymes such as peroxidase,haptens such as digoxigenin, fluorescent dyes or substances capable ofelectro-chemiluminescence or chemiluminescence.

The invention is further elucidated by the following examples.

EXAMPLE 1 Selection of Donors for the Isolation of B Lymphocytes

In order to increase the probability of a successful transformation ofanti-IA-2-specific B lymphocytes from peripheral blood, donors wereselected for the lymphocyte isolation which had a high serum antibodytitre against IA-2.

The antibodies were determined by an in vitro translation assay (seeZhang et al. 1997, Diabetes 46, 40-43 and Dittler J. et al 1998,Diabetes 47, 592-597). Blood was withdrawn from newly diagnoseddiabetics and serum was obtained by known methods. A volume of 2-5 μl ofthe individual sera was incubated overnight at 4° C. with the IA-2icpolypeptide that was radioactively labelled (ca. 15,000 cpm) by in vitrotranslation in 50 μl precipitation buffer (20 mM Tris-HCl, pH 7.5, 150mM NaCl, 1% Triton X-100, 0.1% aprotinin) while rotating. Subsequently50 μl of a 50% protein A-Sepharose suspension was added and it wasincubated for a further hour. Afterwards it was washed three times withthe incubation buffer and the radioactivity of the beads was determinedin a liquid scintillation instrument. A high titre diabetic serum whichproduced ca. 1000 cpm in the immunoprecipitate for a serum quantity of 5μl was used as an arbitrary standard. This value was defined as beingequivalent to 100 U. On this basis the normal sera had levels of ca. 5U. Only lymphocytes from patients whose sera had a titre of more than 80U were used for the subsequent transformation of the peripheral bloodlymphocytes.

EXAMPLE 2 Cell Separation and EBV-transformation

Only those lymphocyte donors whose sera had a titre of at least 80 Uwere used to isolate B lymphocytes. 20-50 ml blood was collected fromthese donors and used to isolate the peripheral mononuclear cells(PBMNC) by means of density gradient centrifugation.

The classical tests for detecting IA-2-specific serum antibodies useprotein A-Sepharose to separate the immune complexes. Hence it must beassumed that the anti-IA-2 antibodies are almost exclusively of the IgGimmunoglobulin class (Zhang et al., Diabetes, 1997, 46, 40-43 andDittler J. et al. Diabetes, 1998, 47, 592-597). However, since the Blymphocytes of peripheral blood produce predominantly IgM, the membraneIgG-positive B cells were isolated in order to enrich the relevant Bcell subpopulation. For this the PBMNC were adjusted to a concentrationof 3*10⁶ cells/ml with ice cold IMDM/10% foetal calf serum (IMDM/10%FCS). Subsequently an anti human-IgG antibody from the mouse was addedat a concentration of 10 μg/ml. The cells were then rolled for 30minutes at 4° C. to prevent the cells from sedimenting during theincubation. They were subsequently centrifuged at 200 * g for 10 minutesat room temperature, the supernatant was aspirated and the cells werewashed twice with IMDM/l10% FCS.

Afterwards the cells were taken up in IMDM/10% FCS at a concentration of1*10⁷ cells/ml (total cell count ca. 1*107 cells) and incubated withMagnetobeads (Dynal M-280) which had been coated with sheep anti-mouseIgG.

Approximately 10 beads per target cell were added (it was assumed thatabout 5% of the PBMNC express membrane IgG).

The cells were rolled with the beads for 30 minutes at 4° C. Afterwardsthe reaction vessel was placed for 5 minutes in the magnetic holder inorder to separate the labelled cells. The supernatant was aspirated, thebeads were resuspended in 1 ml medium and again placed for 5 minutes inthe magnetic holder. The supernatant was again aspirated, the tube wasremoved from the magnetic holder and the isolated cells were resuspendedin 0.5 ml IMDM/10% FCS.

Subsequently 2 ml concentrated Epstein-Barr virus suspension was added.The virus suspension was obtained from the supernatant of a confluentculture of the B 95-8 marmoset cell line (ATCC CRL 1612). The B cellswere incubated for 2 hours in an incubator at 3720 C., 7% CO₂ for thevirus absorption. The tube was moved several times during the incubationphase in order to prevent cell sedimentation.

Afterwards a serial dilution of the separated B cells was made such that100, 200 and 400 B cells were present in 100 μl IMDM/10% FCS. Thenallogenic PBMNC (without CD8-positive cells) that had been irradiatedwith 4000 rad were added to these cell suspensions as feeder cells(50,000 feeder cells per 150 μl cell suspension). Subsequently 100 U/mlIL-6 was added. 150 μl cell suspension per well was aliquoted into96-well round bottom plates and incubated for 2 weeks at 5% CO₂ and 37°C. The medium was changed after 7-10 days.

EXAMPLE 3 Screening Assay for EBV-transformed B Cell Lines that produceIA-2 antibodies

After 2 weeks the culture supernatants of the EBV lines were tested inan ELISA for reactivity to recombinant IA-2. The intracellular part ofIA-2 (IA-2ic) was expressed as the antigen in Escherichia coli incombination with a biotin-labelled peptide at the NH₂ terminus. Thefusion protein was purified by affinity chromatography on a streptavidincolumn.

Streptavidin coated microtitre plates were coated for one hour at roomtemperature with IA-2ic biotin at a concentration of 100 ng/ml.Subsequently the plates were washed with 0.15 mol/l NaCl/0.05% Tween 20.50 μl RPMI/10% FCS was added first to the plates and subsequently 50 μlculture supernatant of the EBV-transformed B cells was added. It wasincubated for 1 hour at room temperature while shaking. Afterwards theplates were washed and 100 μl of a peroxidase-labelled sheep anti-humanFcγ antibody (Boehringer Mannheim GmbH, catalogue No. 1089 196, 100mU/ml in PBS/0.5% bovine serum albumin) was added to detect boundanti-IA-2 antibodies. Subsequently is was again incubated for 1 hour atroom temperature while shaking. Excess conjugate was removed by washingthree times with 0.15 mol/l NaCl/0.05% Tween 20. Subsequently 100 μlABTS® (1 mg/ml, Boehringer Mannheim GmbH, catalogue No. 756 407) in 40mmol/l citrate buffer pH 4.4 containing 3.25 mmol/l sodium perborate wasadded and the absorbance was measured at 405 nm after incubating for 45minutes at room temperature while shaking.

In a typical transformation mixture 2-5*10⁵ membrane IgG-positive Bcells were isolated from initially 1*10⁷ PBMNC. These were divided intoca. 1000 wells. The number of wells that were identified as positive inthe screening test was in the range between 1 and 3 wells per 1000tested wells. The absorbance of the positive wells was in the range1500-2000 mA.

EXAMPLE 4 Cloning EBV-transformed B Cell Lines

Those EBV-transformed B cell lines whose culture supernatant reactedpositive in the ELISA were cloned. For this the cells were depositedsingly into 96-well microtitre plates with the aid of afluorescence-activated cell sorter and irradiated CD8-depleted PBMNC(5*10⁴ cells/well, 4000 rad) were added. The cloning medium was composedof IMDM/10% FCS/100 U/ml IL-6. The culture supernatants containinggrowing clones were tested by means of ELISA and the positive cloneswere expanded. Mass culture for isolating antibodies was carried out ina Tecno mouse bioreactor. The antibodies were isolated from thesupernatant by ammonium sulfate precipitation and chromatography overprotein A or protein G Sepharose.

1. A human monoclonal antibody that binds specifically to islet cellantigen IA-2 in a manner equivalent to that of an antibody from cellline IA-2, 96-3-1, deposit number DSM ACC2365.
 2. The antibody of claim1, wherein said antibody belongs to the immunoglobulin class IgG.
 3. Theantibody of claim 2, wherein said antibody belongs to theimmnunoglobulin subclass IgG1.